1. Field of the Invention
This invention relates to improvements in the art of quantitating radioisotopes whose decay culminates in a production of multiple gamma-photons, and the quantitation of molecules with these isotopes incorporated. The order of magnitude improvements result from an appropriate choice of isotopes and novel apparatus, a Coincident Gamma-photon Detector (CGD), which together achieve an excellent rejection of background radiation events. With the greatly reduced background counts there are advantageous usages of several isotopes which have not previously been utilized for molecular quantitations.
2. Prior Art
The following patents and application describe various known apparatus and methods of detection of radiation.
______________________________________ Name Date Number ______________________________________ Kalish 3-16-76 U.S. Pat. No. 3,944,832 Wilkinson 5-4-76 U.S. Pat. No. 3,954,739 Blumberg et al 2-19-80 U.S. Pat. No. 4,189,464 Kaul et al 11-25-80 U.S. Pat. No. 4,235,864 Nickles 12-23-86 U.S. Pat. No. 4,631,410 Mullani 2-10-87 U.S. Pat. No. 4,642,464 Wong 3-3-87 U.S. Pat. No. 4,647,779 Curtiss et al 6-30-87 U.S. Pat. No. 4,677,057 Karcher et al 6-14-88 U.S. Pat. No. 4,751,389 Ginsberg et al 4-11-89 U.S. Pat. No. 4,802,505 East German (Abstract) 241,788-A ______________________________________
Radioisotopes are detected through the absorption of the energies of decay products. Scintillators are often used to convert the energy of the emitted particle into a burst of low energy photons, which are collected by photodetectors. More specifically low cost organic scintillators (plastic or liquid) are used in prior-art instruments. Their advantages are low cost, a capacity to use complex shapes and fast timing. The major disadvantages excluding their use in the CGD are low gamma stopping power and mediocre energy resolution. Signal amplification and analysis commonly precede the final registration of a decay count.
The sources of background radiation which trigger radiation counters include cosmic rays, radon gas and the traces of man-made and natural radioisotopes contaminating many materials used in radiation counters. Highest in the latter category are carbon-14 and potassium-40. The background of registered counts without a sample present sets a minimum for the amount of radioisotope which can be detected or must be utilized to achieve a valid quantitative assay. There must be enough sample radioactivity to achieve a statistically significant sample count rate over that of the background count. The backgrounds registered by current commercial instruments are in the range of 15-60 counts per minute.
The sensitivity of an assay is thus improved by any measures which reduce background counts. With increased sensitivity shorter counting times and/or reduced amounts of a sample will suffice for a radioisotopic assays. There will be corresponding increases in sample throughput, decreased radiation hazards and less radioactive waste to dispose of.
There is considerable prior art for the reduction of system background counts. Shielding the sample chamber and critical detector components from exterior radiation is a common measure. Very pure shielding materials are used to minimize their contribution of contaminating radioisotopes. Parameters of detection systems can be set to reject background events falling outside of the energy window(s) characteristic of the emissions of a known isotope. For example gamma emitting isotopes have nuclei which drop to a lower angular momentum state(s) with emission of a monoenergetic gamma-photon. In a typical gamma counter, the scintillator converts the gamma to a burst of lower energy photons which are absorbed by a photodetector and the energy quantitated with associated electronics. Energy depositions outside of the energy window of the isotope's gamma are not counted. To recognize background due to cosmic rays, a detector external to the sample chamber shield can be used in conjunction with the sample chamber's instruments. The external veto counter rejects a coincident count from the sample chamber.
Using these prior-art techniques a very low background apparatus has been constructed which reduces backgrounds to a few counts per hour. To achieve backgrounds of a few counts per day, the detectors are placed deep underground. The state of the art in ultra-low background counting is represented for example in reports of R.L. Brodzinski et al.. NIM A239, (1985) 207. and R.L. Brodzinski et al., "Further reduction of radioactive backgrounds in ultrasensitive germanium spectrometers," NIM, in press. Only three such instruments now exist worldwide because the system costs more than $1,000,000. They are far too costly for routine molecular quantitation tasks. Moreover they are not optimized for particular isotopes and are not designed for the high throughput needs of the chemical, biological of commercial diagnostic laboratories.
3. Invention Development
In the development of this invention, information was gathered with one of the ultra-low background systems. A particular interest was the background intrinsic to body fluids. Assaying biological macromolecules is a projected major area of application of this invention. With a 50 ml blood sample, it was observed with the gamma counting instrumentation that:
a) the background increases rapidly for low energies, E .ltoreq. 300 keV (kilo electron volts), with indications of a few discrete lines with count rates of a few hundred counts per hour; PA1 b) the background is a few counts per hour in the range 300 .ltoreq. E .ltoreq. 500 keV; PA1 c) there is a 511 keV peak of a few tens of counts per hour, attributable to positrons in the sample producing 511 keV annihilation gamma pairs; PA1 d) the background in the range 600 .ltoreq. E .ltoreq. 1000 keV was below a count per hour. PA1 e) there is a background attributable to potassium-40 with count rates of the order of 100 counts per hour. PA1 1. There is coincident activation of three gamma detectors within an interval compatible with the known temporal statistics of the isotopic decay: typically an interval of about 10 nanoseconds and generally much less than 100 nanoseconds; PA1 2. The gamma energies are compatible with those of the sample's isotope comprising two 511 keV annihilation gammas and that of the solitary gamma(s) emitting during the transition in angular momentum state of the daughter nucleus; PA1 3. The angular distribution of the three candidate gammas is compatible with that of an opposed 511 keV annihilation pair, and the third directionally-noncorrelated gamma.
Background can be rejected by the use of time coincidence methods, as achieved for example in prior-art uses of positron emitting isotopes. This decay signature is used in the rejection of single gamma background events, as employed for example in positron emission tomography. However, the evident presence in blood of significant traces of positron emitting isotopes set an undesirable high background for rejections employing only double coincidence.
Advantage can be taken of the existence of isotopes with more complex decay signatures. There are isotopes whose decay with the concurrent production of more than two gamma-photons. Among them there is a substantial sub-family which initiate decay through the emission of a positron and leave the nucleus in an excited angular momentum state, leading to prompt gamma emission. This solitary gamma plus the two annihilation photons (E = 511 keV) derived from a positron-electron interaction yields a triple of coincident gammas with known energies. Thus the net decay signature is a production of the back-to-back 511 keV gamma pair and a solitary gamma with a non-correlated emission direction and distinct energy (for some of the isotopes the solitary gammas can have a few different energies). The average delay between the appearance of the annihilation pair and the solitary gamma generally is much less than 100 nsec (nanoseconds). Typically it is about 0.1 nsec within liquids or solids and 10-100 nsec in air at one atmosphere of pressure.
For brevity, members of this family will be termed "triple gamma isotopes." As chemical reagents they include carbon, nitrogen, oxygen, fluorine, bromine and iodine. The chemistry of iodine is particularly useful. Through simple adduction at double covalent bonds such as &gt;C=C&lt; and -N=C&lt;, iodine is used to radioisotopically label preformed macromolecules including ribonucleic acids, deoxyribonucleic acids, carbohydrates and proteins. The single-gammaemitters iodine-125 and iodine-131 are extensively utilized to label antibodies and/or antigens for the radioimmunoassay (RIA) procedures of biomedical research and medical diagnostics. Immunoassays utilize the exquisite binding specificities of antibodies to quantitate either antigens or antibodies, and the quantitations can proceed in complex body fluids or on tissue samples. RIA is the most sensitive of the immunoassay techniques. Methodologies are well described in: A.E. Botton, W.H. Hunter, "Radioimmunoassay and related methods," page 26.1-26.55 in the Handbook of Experimental Immunology, ed. L.A. Herzenberg et al., publisher Blackwell Scientific, 1984 and D. Freifelder. "Physical Biochemistry". chpt. 10, publisher W.H. Freeman, 1977, D. Bereitag, K.H. Voigt. in Treatise on Analytical Chemistry, Part I, p. 285- 333, publisher J. Wiley & Sons. Triple gamma emitting isotopes have not been previously utilized for RIA procedures, and more generally, for sensitive quantitations of molecules.
The sources of background affecting the quantitation of triple gamma isotopes was explored with pilot instrumentation. A three detector assembly was used. Each detector had a NaI(Tl) scintillator coupled to two inch photodetectors, Ortec preamplifiers and spectroscopy preamplifiers, appropriate signal delay lines and a high voltage power supply. No external shielding was employed. One detector served as a master to initiate a coincidence interval of 50 nsec. Three coincidence counting modes were implemented: (1) to count all events registered by the master; (2) to count a master event if accompanied by a coincident event in another counter; and to count a master event if accompanied by coincident events in the two other detectors. In this third mode, two of the energy windows were 511.+-.50 keV. The energy spectrum was measured for each coincidence mode using multichannel analyzers.
For the experiment of FIG. 1a the 50 ml liquid sample was blood. This trial represents a worst case bioassay situation because of the presence of the single-gammaemitting potassium-40 in biological fluids and tissues. A major projected use of the invention is the sensitive quantitation of biological macromolecules in the presence of body fluids. With no coincidence requirement the energy spectrum reflects the background, dominated by potassium-40 gammas and their lower energy Compton scattering events. With the double coincidence requirement, the background count is reduced about 100 fold. These counts are attributed predominantly to traces of positron emitting isotopes. The triple coincidence mode corresponds to a selective acceptance condition for triple gamma producing isotopes. The accepted count is decreased about another 50 fold, as compared to the dual coincidence mode.
The peak at 1550 keV is attributed to 510 keV gammas from ubiquitous radon gas which are accidently coincident with annihilation gamma pairs derived from traces of positron emitting isotopes, as 510+2.times.511=1532 keV. This peak doubled in height when air with its radon replaced 50 ml of blood or urine during acquisition of a triple coincidence spectrum, as shown in FIG. 1b. With the air sample an increasing background towards lower energies is attributed to Compton scattered gammas, which contribute to accidental gamma triples arriving within the 50 nsec coincident interval. This contribution is quenched by the presence of more absorbing blood or urine.
This proof of concept experiment illustrates that excellent background rejection can be achieved with a CGD, when only events indistinguishable from a triple gamma signature are counted. These results and those obtained with the ultra-low background system guided design of this invention.
The experimental results also guide choices of triple gamma isotopes most suitable for radioisotopic labeling of molecules. Preferably, the solitary gamma should have an energy distinguishable from the 510 keV radon-222 gamma, so that the gamma background from radon can be most effectively rejected. Furthermore, the energy spectrum from blood obtained with the ultra-low background counter recommends an energy greater than 300 keV and preferably in the 600-1000 keV range. A survey of over 1000 known isotopes was made for triple gamma isotopes which satisfied the above criteria. Also a halflife of at a least a few hours is desirable to accommodate production, radioisotopic labeling chemistries and distribution to users. Several suitable isotopes are thus available:
______________________________________ Isotope halflife E in meV of solitary gamma(s) ______________________________________ selenium-137 7.1 hours 1.31, 0.86 bromine-76 17 hours 1.21, 0.75, 0.33 germanium-69 40 hours 0.576 bromine-77 57 hours 0.813, 0.520, 0.237 iodine-124 4.2 days 0.72, 0.6 iodine-126 13 days 0.64, 0.395 ______________________________________
The iodine isotopes with their longer halflives and simple adduction chemistries are particularly well suited for use as radioisotopic labels for other molecules. The preferred embodiment of the CGD is optimized for iodine-124 quantitation. Among the bromine and iodine isotopes the best background rejection can be achieved with the highest energy E = 0.72 keV gamma of iodine-124.
The preferred embodiment of the CGD is optimized to the needs of the RIA with iodine isotopes. Quantitations will be feasible much below the level of the backgrounds of contemporary assay systems. Thus diagnostic detections of antigens (such as cancer or HIV virus indicators) will be much more sensitive.
More generally assays of the molecules incorporating the triple gamma isotopes can be performed with much higher sensitivity. At least a thousand fold reduction in the minimal amount of radioisotope necessary for an assay will be achievable with CGD instruments, and corresponds to less than a nanocurie of triple gamma isotope. The corresponding amount of radiation is less than that from radioactive contaminants of television screens or the drinking water in areas of the Rocky Mountains.
There is a novel application area. With the minimal necessary quantity of triple gamma isotope for assays performed in body fluids, the amount of energy deposited by a triple gamma isotope is much less than that of the resident radioisotopes, primarily potassium-40. Thus when desirable certain biochemical reaction component of an assay could be performed within the body, with an insignificant added radiological burden to the organism/patient. Subsequently the appropriately mounted sample would be withdrawn for radioisotopic quantitation in a CGD. For a single example, an antibody for the AID (or HIV) virus would be coupled to the surface of a flexible, thin plastic rod or ribbon. The binding of its conjugate iodine-126 labeled viral antigen would prepare the rod with its antibody-antigen complex for a displacement assay. When inserted into a blood vessel or body cavity, the disassociation of the iodine-126-antigen from the antibody would be accelerated, by the presence of homologous antigen competing for the two binding sites of each antibody. After a chosen interval, the plastic would be withdrawn and its retained iodine-126 antibody quantitated in a CGD. From the retention, time in body fluid and calibration parameters, the concentration of the viral antigen in the body fluid would be calculated. The great value in such in situ assays would be avoidance of numerous artifacts which can accompany removal of biological specimens from their natural environment.
It is evident that quantitations utilizing a CGD are entirely distinct from usages of positron emitting isotopes in the imaging applications of Positron Emission Tomography (PET). A CGD will be a compact instrument, accommodate micro-samples, and be suitable for isotopes with long half lives. PET systems occupy a few rooms, are designed to accommodate people and require very short lived isotopes. A CGD assay will require about a nanocurie of isotope while PET imaging runs require tens of millicuries of isotope. The CGD uses only several scintillators while PET requires hundreds. The difference in the channels of required electronics leads to different technological challenges and design trade offs.